A: Time course of hematocrit change after subcutaneous insertion of empty or testosterone implants in female mice (*p=0.0002, **p<0.0001, ***p=0.0002 for between-group comparison at each time). The slope of hematocrit change over time was 0.65 for testosterone group (p<0.0001) and 0.02 for control group (p=0.6121).B: The mice treated with testosterone implants for 2-weeks had significantly higher hemoglobin (Hgb), hematocrit (Hct), and a trend towards higher mean corpuscular hemoglobin (CHm) than mice that received empty implants.C: Testosterone-treated mice had significantly higher serum iron, lower total iron binding capacity (TIBC), and higher transferrin (Tf) saturation than controls 2 weeks after insertion of either empty (C) or testosterone implants (T).D. Testosterone-treated mice (T) had significantly higher reticulocyte count (Retic-C) and higher reticulocyte hemoglobin ratio (CHr) than controls (C) after 2 weeks of treatment.The data are mean±SEM, n=10-20 mice for each measurement.

A. Western analysis of splenic ferroportin (fpn) expression (N=5 for each group). Ferroportin protein level, normalized by actin expression, was significantly higher in spleens of testosterone-treated (T) mice than in controls (C).B. Prussian blue staining for iron in the spleen of mice treated with either empty (C) or testosterone-containing (T) implants (N=3 for each group). Ferric iron stains greenish-blue and background tissue is stained with hematoxylin. Testosterone treatment significantly reduced splenic ferric iron, measured either as area of iron staining (upper left panel) or as the size of iron stains (lower left panel). This result is consistent with the increased expression of iron-exporter ferroportin in this tissue.C. Iron incorporation into red blood cells. Female mice were injected testosterone twice weekly for 2 weeks. 58Fe/transferrin complex was injected into tail vein at 10 ng/g body weight. After 8 h, blood was taken to measure 58Fe/56Fe ratio using MC-ICPMASS. The amount of iron incorporated into red cells was calculated from specific activity of 58Fe in steady-state serum iron pool size.D. Effects of testosterone on hemoglobin accumulation in K562 cells. Erythroid differentiation was induced by sodium butyrate (0.5 mM) for 48 h. Cells were incubated with 10% serum from female mice pre-treated with vehicle (C) or testosterone (T). Serum iron concentrations were 17.5±0.9uM and 25.1±0.6uM and transferrin saturation 31.0±0.7% and 36.7±0.3% for the control and T-treated mice, respectively. A negative control was incubated with serum-free medium (N). Cells were harvested after 24 h and stained with benzidine (upper panel). Cells were lysed with 0.2% Triton and hemoglobin was measured after benzidine staining at OD600.

Erythropoietin is not essential for testosterone-mediated suppression of hepcidin

A: Time-course of testosterone-induced changes in renal EPO mRNA expression (upper panel) and serum EPO concentrations (lower panel) in female mice. Results are mean±SEM, N=8 for each group. Testosterone administration induced a rapid increase in renal EPO mRNA expression which was followed by an increase in serum EPO levels.B: To determine whether EPO is the mediator of testosterone-induced hepcidin suppression, we treated female mice with testosterone (T) with and without an anti-EPO neutralizing antibody (anti-EPO). EPO and hepcidin mRNA expression levels were assessed 72h (left panel) and 2 weeks (right panel) after treatment initiation. Testosterone administration was associated with a 2 fold increase in renal EPO mRNA expression at 72 h and ~75% decrease in hepatic hepcidin mRNA expression. The administration of anti-EPO antibody alone resulted in a 2 fold increase in renal EPO mRNA. Combined administration of testosterone and anti-EPO antibody resulted in greater increase in renal EPO mRNA expression than either intervention alone. The administration of anti-EPO antibody resulted in a nearly 2 fold increase in hepatic hepcidin mRNA expression. However, co-treatment with anti-EPO did not prevent testosterone-induced suppression of hepcidin either at 72h or 2 weeks. Thus, EPO is not essential for mediating the inhibitory effects of testosterone on hepcidin expression. Results are mean±SEM, N=5 for each group.

Androgen receptor associates with Smad1 and Smad4, blocks BMP/Smad signaling, and reduces Smad1 binding to the BMP-response elements (BMP-RE1 and BMP-RE2) in the hepcidin promoter

A: Testosterone administration for 48h upregulated the expression of androgen receptor (AR) and phospho-Smad1, but had no effect on the expression of total Smad1 and Smad4. The results are representative of 3 experiments. Each lane represents the result from one animal.B: Liver nuclear extracts were immunoprecipitated with goat-anti-Smad4 (left panel) or rabbit-anti-AR (right panel) antibodies. Immune complexes were separated by gel electrophoresis and detected using anti-Smad1, anti-Smad4, anti-CBP/p300, and anti-AR antibodies. Immune complexes immunoprecipitated with anti-Smad4 antibody contained AR, Smad1 and p300 proteins (left panel). Similarly, Immune complexes immunoprecipitated with anti-AR antibody contained Smad4, Smad1, and p300 (right panel). All samples were pre-cleared with agarose gel conjugated with normal goat IgG (for ip with Smad4) or rabbit IgG (for ip with AR) before first antibody was added to the reaction. Equal inputs were confirmed by Western blot for LaminA/C (not shown). Results are representative of 4 experiments. These data provide evidence of association of AR with Smad1, Smad4, and CBP/p300.C: Liver hepcidin mRNA expression in mice treated with vehicle (C), dorsomorphin (DM) (an inhibitor of BMP/Smad1 signaling), DM plus testosterone (DM+T), or T alone. Administration of DM and T each down-regulated liver hepcidin mRNA expression. However, combination of both did not decrease it any more than T alone, suggesting that T and DM likely share overlapping pathways for hepcidin regulation. Results are mean± SEM, N=4 for each group.D: Liver tissue isolated from mice treated with vehicle (C) or testosterone (T) was subjected to ChIP analysis. Immuno-precipitation of Smad1 protein-DNA complexes was performed using anti-Smad1 (Cell Signaling#6944). Negative controls (Neg) were immuno-precipitated with rabbit IgG and positive controls (Pos) were immuno-precipitated with rabbit anti-histone 3 (H3). Real-time PCR was performed using primer sets flanking BMP-RE1 and BMP-RE2 of mouse hepcidin promoter. Results were normalized to the corresponding inputs (sonicated chromatin before immuno-precipitation). Treatment with testosterone for 48h reduced the association between Smad1 and the BMP-RE1 (left panel) and BMP-RE2 (middle panel) on the hepcidin promoter. The assay specificity was validated by primers designed for RPL3 intron 2 (Cell Signaling, #7015) which strongly binds to H3 but not Smad1 or rabbit IgG (right panel). Results are mean±SEM, N=3 for each group.

A: HepG2 cells were transfected with control vector, low dose (200 ng/well), and high dose (800 ng/well) of AR encoding plasmid, all in combination with a pGL4 luciferase reporter driven by a 3kb wild-type hepcidin promoter (300 ng/well). A CMV-driven Renilla luciferase vector (30 ng/well) was used as transfection control. 12h after transfection, cells were switched to 1% FBS in low glucose DMEM containing graded dose of DHT and flutamide. After 12h, BMP2 (10 ng/ml) was added to selected wells and incubation was extended for 12h. Results were normalized to Renilla luciferase activity. Experiments were repeated 3 times. Data are mean±SEM.B: HepG2 cells were transfected with control vector or AR-encoding plasmid (800 ng/well). The AR-transfected cells were treated with DHT (100nM). BMP2 was added at 0, 25, 50, and 100ng/ml and incubation was extended for 4h. Hepcidin mRNA was analyzed by real-time PCR and normalized to HPRT. Results are mean±SEM, N=4 for each treatment.